Intermolecular dark resonance energy transfer (DRET) for high contrast imaging of endogenous mRNAs in 3D biological samples.

In cellulo detection of RNA molecules is a cornerstone for understanding many biological processes, with recent advances heavily centered on fluorescence-based strategies. The fluorescence resonance energy transfer process stands out as an indispensable tool due to the high specificity of the signal emitted by the acceptor when in proximity to the donor. However, inherent crosstalk between the spectral bands of FRET-compatible fluorophores often diminishes detection sensitivity. Capitalizing on the unique capacity of poorly emissive fluorophores to transfer their energy by resonance, we previously introduced the intermolecular Dark-RET (DRET) concept. This approach has the advantage of minimizing the background noise while maximizing the fluorogenic response via the acceptor emission channel. In this study, we refined both theoretical and hands-on aspects of the DRET approach, providing a readily accessible framework to the scientific community. We further underscored its potential to generate a specific fluorogenic response upon target recognition by detecting the oskar mRNA in the biological context of Drosophila oocytes. Combining in situ hybridization experiments with confocal imaging, we demonstrated the ability of the intermolecular DRET approach to generate high signal-to-noise ratios and specific target recognition, highlighting the benefits of the intermolecular DRET strategy compared to standard FRET approaches.